TRYPTOPHAN FLUORESCENCE IN ELECTRON-TRANSFER FLAVOPROTEIN-UBIQUINONE OXIDOREDUCTASE - FLUORESCENCE QUENCHING BY A BROMINATED PSEUDOSUBSTRATE

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda-ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda-m 334 nm) and the bandwidth (DELTA-lambda-1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda-m or DELTA-lambda-1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (K(m) = 2.6-mu-M; k(cat) = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (K(d) = 1.6-3.2-mu-M) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (K(m) = 2-mu-M; k(cat) = 162 s-1) and the analogue Q(o)(CH2)10OH (K(m) = 2-mu-M; k(cat) = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.